Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
1994-10-28
2001-10-02
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S401000, C435S304100
Reexamination Certificate
active
06297046
ABSTRACT:
TECHNICAL FIELD
This invention relates to multi-layer films, and containers formed therefrom for the in vitro culture of cells. Specifically, the invention is directed to a multi-layer, flexible, gas permeable container having an inner growing surface of polystyrene, which is conducive to the culture of cells.
BACKGROUND ART
There are two major types of cells grown in vitro: suspension cells (anchorage-independent cells); and adherent cells (anchorage-dependent cells). Suspension or anchorage-independent cells can multiply, in vitro, without being attached to a surface. In contrast, adherent cells require attachment to a surface in order to grow in vitro. Additionally, some non-adherent cells grow best on a surface that promotes adherent cell growth.
It is known to grow adherent cells, in vitro, in polystyrene flasks. Polystyrene is the most common type of plastic used in the manufacture of rigid, gas impermeable cell culture flasks or plates. It is thought that polystyrene promotes the growth of adherent cells because of its ability to maintain electrostatic charges on its surface which attract oppositely charged proteins on the cell surfaces. However, to date, the available polystyrene culture containers have been of the rigid flask or plate type because polystyrene is known in the art as a rigid, gas-impermeable plastic.
Cells are commonly cultured in a growth medium within polystyrene or other containers placed in enclosed incubators. In addition to providing a certain degree of isolation from pathogens, the incubators maintain a constant temperature, usually 37° C., and a constant gas mixture. The gas mixture must be optimized for a given cell type, and be controlled for at least two parameters: (1) partial pressure of oxygen (pO
2
) to serve the aerobic needs of the cells, and (2) partial pressure of carbon dioxide (pCO
2
) to maintain the pH of the growth medium. Since the known types of rigid cell culture containers are gas impermeable, their lids or caps are not sealed onto the containers. Rather, they are offset sufficiently to allow gas exchange through a gap or vent between the cap and the container. Such a container is disadvantageous for clinical uses because the vent might allow contamination of the culture or lead to accidents involving biohazardous agents.
In addition to polystyrene flasks, others have constructed flexible, breathable containers for containing adherent cells to be grown in vitro. For example, the commonly assigned U.S. Pat. No. 4,939,151 provides a gas-permeable bag with at least one access port. This allows for a closed system (ie., one without a vent). The bag disclosed in the '151 Patent is constructed from two side walls. The first side wall is made of ethylene-vinyl acetate (“EVA”) which may be positively or negatively charged. The second side wall is constructed from a gas permeable film such as ethylene-vinyl acetate or a polyolefin. The first side wall is sealed to the second side wall along their edges. While EVA can hold an electrostatic charge, the charge has the undesirable tendency to decay over time. Eventually, the decay will render the container ineffective for growing adherent cells.
It has been found that the cell growth rate within a sealed container may be influenced by the gas permeability characteristics of the container walls. The optimal gas requirements, however, vary by cell type and over the culture period. Thus, it is desirable to be able to adjust the gas permeability of the container. The polystyrene flask, and the flexible flask which is entirely constructed from a monofilm, do not provide for such adjustability.
SUMMARY OF THE INVENTION AND OBJECTS
The present invention provides a multi-layer, co-extruded film suitable for producing gas-permeable cell culture bags. The film has an ultra-thin first layer of polystyrene having a thickness from about 0.0001 inches to about 0.0010 inches. The film has second layer adhered to the first layer made of a polyolefin. The polyolefin acts as a flexible substrate for the polystyrene to provide a flexible, gas permeable film. Thus, the second layer is sometimes referred to as the substrate layer.
The film may also have an adhesive tie layer interposed between the first and second layers. The film may also have one or more additional outer layers of polyolefin (such as polypropylene) to provide strength and scratch resistance, as well as additional tie layers interposed between these additional layers.
The film most preferably has the following physical characteristics: (1) a mechanical modulus of between about 10,000 and 30,000 psi (ASTM D 790); (2) an oxygen permeability within the range of about 9-15 Barrers; (3) a carbon dioxide permeability of 40-80 Barrers; (4) a nitrogen permeability of 10-100 Barrers, and (5) a water vapor transmission rate of not more than 20 (g mil/100 in
2
/day). Preferably, the film should have an optical clarity of between about 0.1% to about 10% as measured by a Hazometer in accordance with ASTM D1003.
The present invention also provides a flexible, gas-permeable cell culture container constructed from the above described films, with the polystyrene layer forming the inner surface of the container.
Another aspect of the present invention provides a flexible, gas-permeable cell culture container whose gas permeability may be adjusted to best match the requirements of the cell being cultured in the container. The multi-layer structure of the present film allows one to vary the material of the second layer or substrate layer and its thickness to achieve the desired or predetermined gas permeability requirements for cell growth. Preferably, the type and thickness of the substrate layer will be selected to optimize cell growth.
Another aspect of the invention provides for various embodiments of culture containers some of which are advantageous for growing adherent cells, non-adherent cells, and both.
Another aspect of the invention is to provide a flexible, gas permeable cell culture container having a first side that is suitable for growing adherent cells, a second side for growing non-adherent cells, and indicia on the container for indicating the first side from the second side.
REFERENCES:
patent: D. 285725 (1986-09-01), Franchere
patent: 2705223 (1955-03-01), Renfrew et al.
patent: 2990306 (1961-06-01), Dyer
patent: 3255923 (1966-06-01), Soto
patent: 3375300 (1968-03-01), Ropp
patent: 3419654 (1968-12-01), Chiba et al.
patent: 3485782 (1969-12-01), Nagle
patent: 3589976 (1971-06-01), Erb
patent: 3655503 (1972-04-01), Stanley et al.
patent: 3661674 (1972-05-01), Higgs et al.
patent: 3772136 (1973-11-01), Workman
patent: 3885081 (1975-05-01), Van Paesschen et al.
patent: 3912843 (1975-10-01), Brazier
patent: 3937758 (1976-02-01), Castagna
patent: 3960997 (1976-06-01), Sorensen
patent: 3995084 (1976-11-01), Berger et al.
patent: 4005710 (1977-02-01), Zeddies et al.
patent: 4041103 (1977-08-01), Davison et al.
patent: 4045515 (1977-08-01), Isaka et al.
patent: 4058647 (1977-11-01), Inoue et al.
patent: 4085244 (1978-04-01), Stillman
patent: 4087587 (1978-05-01), Shida et al.
patent: 4087588 (1978-05-01), Shida et al.
patent: 4095012 (1978-06-01), Schirmer
patent: 4103686 (1978-08-01), LeFevre
patent: 4112989 (1978-09-01), Grode et al.
patent: 4140162 (1979-02-01), Gajewski et al.
patent: 4147827 (1979-04-01), Breidt, Jr. et al.
patent: 4156709 (1979-05-01), Kondo et al.
patent: 4161362 (1979-07-01), Blake
patent: 4188350 (1980-02-01), Vicik et al.
patent: 4226822 (1980-10-01), Yoshikawa et al.
patent: 4227527 (1980-10-01), DeFrank et al.
patent: 4230830 (1980-10-01), Tanny et al.
patent: 4233367 (1980-11-01), Ticksor
patent: 4244378 (1981-01-01), Brignola
patent: 4261473 (1981-04-01), Yamada et al.
patent: 4274900 (1981-06-01), Mueller et al.
patent: 4286628 (1981-09-01), Paradis et al.
patent: 4294935 (1981-10-01), Kodera et al.
patent: 4310017 (1982-01-01), Raines
patent: 4311807 (1982-01-01), McCullough, Jr. et al.
patent: 4322465 (1982-03-01), Webster
patent: 4322480 (1982-03-01), Tuller et al.
patent: 4327726 (1982-05-01), Kwong et al.
Bacehowski David V.
Bender James G.
Kolanko William
Ling Michael T. K.
Rosenbaum Larry
Baxter International Inc.
Beisner William H.
Buonaiuto Mark J
Fuchs Joseph A
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